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Methylation Interference Assay
The methylation interference assay is an analytical method that is used to determine which nucleotides in a DNA molecule are important for protein binding(1). This method provides information on where protein binding sites are and what proteins bind to a specific DNA molecule. Thus providing insight into how a gene may be regulated. Technique Initially DNA fragments are 32p end labeled after which guanine and adenine nucleotides are methylated by treatment with dimethyl sulfate. Treatment with this chemical generally results in 1 modified base per DNA molecule (1). Next the protein of interested is incubated with the methylated DNA fragment. If the DNA is modified at nucleotides that are involved in protein binding, the protein will be unable to bind to the DNA. However, if methylation has occurred at nucleotides that are not important to protein binding, the protein will be able to bind to the DNA (1). DNA fragments that are bound by protein and those that are not bound by protein can be separated via electrophoretic mobility shift assay. Other common techniques used to separate bound and unbound DNA fragments include filter binding and immunoprecipitation (1). Next the DNA molecules are treated with an enzyme, such as piperidine, which will cleave the DNA molecules into smaller fragments. Piperidine cleaves DNA at modified bases. Finally the fragments are run on a denaturing gel along with a sequencing ladder. Because each DNA molecule is only methylated at a single position the sequencing ladder can be used to determine which nucleotide had been modified. Cleavage fragments that are generated from unbound DNA will differ from the fragments generated from DNA that was bound by protein. Thus the fragments generated from bound or unbound DNA can be used to determine the nucleotides that are important in protein binding. History and Significance The methylation interference assay was developed from the Maxam-Gilbert DNA sequencing method (1) which also uses the methylation of guanine and adenine nucleotides followed by cleavage of these modified residues by piperidine (2). This technique was originally used to determine which factors were involved in modulating expression of the immunoglobin gene (3) but has been used to study a wide range of problems from disease susceptibility (4) to muscle function (5). It is especially useful due to its ability to resolve single nucleotides and is one of the highest resolution methods used to investigate sequence specific DNA-protein interaction (1). Sources 1. Carey M, Smale ST Methylation interference assay. Cold Spring Harbor Protocols, 2007. 2. Maxim AM, Gilbert W. A new method for sequencing DNA. PNAS. 1977; 74: 560-564. 3.Ephrussi A, Chuch GM, Tonegawa S, Gilbert W. B-lineage-specific interactions of an immunoglobulin enhancer with cellular factors in vivo. Science. 1985; 227: 134-140. 4. Baiping Z, Shu Y, Herrmann SM, Eriksson P, Maat M, Evans A, Arveiler D, Luc G, Cambien F, Hamsten A, Watkins H, Henney A. Functional polymorphism in the regulatory region of gelatinase B gene in relation to severity of coronary atherosclerosis. Circulation. 1999; 99: 1788-1794. 5. Lassar AB, Buskin JN, Lockshon Dm Davis RL, Apone S, Hauschka SD, Weintraub H. MyoD is a sequence-specific DNA binding protein requiring a region of myc homology to bind to the muscle creatine kinase enhancer. Cell. 1989; 58(5): 823-831. 6. https://www.nationaldiagnostics.com/electrophoresis/article/methylation-uracil-interference-assays